Selection and screening in tissue culture for resistance of plant varieties to fungal pathogens is known to the art. Helgeson JP (1983) in Use of Tissue Culture and Protoplasts in Plant Pathology, eds. Helgeson JP and Deverall BJ, pp. 9-38, reviews this subject. Examples include resistance of potato (Solanum tuberosum) to Phytophtora infestans (Behnke M (1979) Theor. Appl. Genet. 55:69-71; Behnke M (1980) Theor. Appl. Genet 56:151-152) and to Fusarium oxysporum (Behnke M (1980) Z. Pflanzenzuchtg 85:254-258), resistance of maize (Zea mays) to Helminthosporium maydis race T (Gengenbach BG and Green CE (1975) Crop Sci. 15:645-649; Gengenbach BG et al. (1977) Proc. Natl. Acad. Sci. USA 74:5113-5117), resistance of rape seed (Brassica napus) to Phoma lingam (Sacristan MD (1982) Theor. Appl. Genet. 61:193-200 and 63:96), and resistance of soybeans Glycine max) to Phytophthora megasperma var. sojae (Holliday MJ and Klarman WL (1979) Phytopathol. 69:576-578). Haberlach GT et al. (1978) Plant Physiol. 62:522-525, showed that addition of cytokinins to the culture medium resulted in an improved differential response in culture of tobacco (Nicotiana tabacum) calli derived from resistant and susceptible plants to Phytophthora parasitica var. nicotianae race 0. However, they did not note a correlation between the improved differential response and the greening of the calli.
The art recognizes that resistance in culture (in vitro) and in vivo may not be correlated in all pathogen/plant systems. A genotype having in an intact plant a phenotype of resistance might be sensitive in culture. Conversely, a resistant callus might regenerate into a susceptible plant (see Helgeson, supra; Scowcroft WR et al., in Helgeson and Deverall, supra; Keen NT and Horsch R (1972) Phytopathol. 62:439-442). For instance, physiological responses specific to a particular tissue, organ, or state of differentiation may be different in plants than in cultured cells. Similarly, anatomical barriers present in plants are absent in cultured tissues. It is also recognized in the art that levels of gene expression in vivo and in vitro can be very different. For instance, Widholm J (1980) in Plant Cell Cultures: Results and Perspectives, eds: Sala F et al., provides an example of a gene specifying resistance to a toxic chemical which is expressed differently in cultured cells than in plants. If a correlation can be made between action of the pathogenic agent in vitro and in vivo the behavior of a pathogen/plant system becomes predictable from in vitro bioassays. The present invention teaches such a correlation.
Brown stem rot is a disease of soybeans caused by the fungus Phialophora gregata, which has often been identified in publications as Cephalosporium gregatum (Allington WB and Chamberlain DW (1948) Phytopathol. 38:793-802). Brown stem rot is a major pest of soybeans (e.g. see Seim D (Mid-Feb., 1985) pp. 24-D to 24-E) and is capable of reducing yields as much as 38% (Gray LE (1972) Plant Dis. Reptr. 56:580-581; Gray LE and Sinclair JB (1973) Plant Dis. Reptr. 57:853-854). In screening of soybean plants for brown stem rot resistance by conventional means, two resistance genes have been identified. There is a resistance/susceptibility gene, having resistance as the dominant allele. A modifier or minor nonallelic gene having resistance to brown stem rot as the dominant allele has also been detected (Sebastian SA and Nickell CD (1985) J. Hered. 76:194-198). Screening for resistance involves wounding and inoculation of the stems of individual plants (e.g. see Gray, supra; Gray and Sinclair, supra) or root inoculation with the fungus (Sebastian SA et al. (1983) Crop Sci. 23:1214-1215).
Isolates of P. gregata can be grouped into two classes, both of which cause vascular browning and one of which, Type I, produces a toxin which causes wilting and defoliation in sensitive soybeans, but not in resistant soybeans (Gray LE (1971) Phytopathol. 61:1410-1411; Gray LE and Chamberlain DW (1975) Phytopathol. 65:89-90). Three compounds, gregatin A, gregatin C, and gregatin D were characterized as causing some symptoms characteristic of brown stem rot on adzuki beans and of additionally inhibiting bacterial and fungal growth at concentrations lower than caused symptoms on plants; gregatin A was reported to give the most characteristic symptoms (Kobayashi K and Ui T (1975) Tetrahed. Lett. 47:4119-4122 and (1977) Physiol. Plant Pathol. 11:55-60). However, gregatin A is also produced by Aspergillus panamensis, which is not a plant pathogen (Anke H et al. (1980) J. Antibiot. 33:931-939). The structure of gregatin A as published by Kobayashi and Ui, supra, and Anke et al., supra, was found to be incorrect and was reassigned by Clemo NG and Pattenden G (1982) Tetrahed. Lett. 23:589-592. Reeder RT (1985) M.S. Thesis, University of Illinois at Urbana, showed that although gregatin A has some inhibitory effect on photosynthetic electron transport, it is not responsible for species-specificity and genotype-specificity in the brown stem rot disease.